The chemiosmotic hypothesis predicts that in bacteria inward movement of hydrogen ions is coupled to ATP synthesis catalyzed by the membrane-bound CA2 ion, Mg2 ion-stimulated ATPase. To test this idea hydrogen ion entry into cells of Streptococcus lactis and Escherichia coli will be studied after imposition of an electrochemical potential difference for H ion (a "protonmotive force") of varying size. The membrane potential will be measured by distribution of K ion in the presence of valinomycin and from the fluorescence of cyanine or oxonol dyes. The pH gradient will be measured by distributions of weak acids and bases, and from the amount of H ion which enters the cell. A study of these parameters in the presence and absence of ATP synthesis will give information about coupling between ATP synthesis and H ion movements. Other experiments will focus on active transport of galactosides by the lactose transport system of E. coli. This system normally couples 1 H ion/substrate molecule during transport. Mutants will be isolated which use sodium rather than the hydrogen ion as the coupling cation. The chemical effect of cation binding on substrate interaction with the transport protein can then be studied. Other mutants will be isolated which accumulate substrates to abnormally high levels. Such mutants may have altered stoichiometry of H ion/substrate coupling.